Production and characterization of semi-quinoloneantibiotic produced by Streptomyces griseorubensWaleed Abdulkhair  ( waleed_hamada@yahoo.com )

National Organization for Drug Control and ResearchMousa Alghuthaymi 

Shaqra University

Research Article

Keywords: antimicrobial, geneticalmethods

Posted Date: December 17th, 2020

DOI: https://doi.org/10.21203/rs.3.rs-121133/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

Page 1 of 16

Production and Characterization of Semi-Quinolone

Antibiotic Produced by Streptomyces griseorubens

Waleed M. Abdulkhair1,* and Mousa A. Alghuthaymi2

1 National Organization for Drug Control and Research (NODCAR),

Microbiology Department, Giza, Postcode 12553, Egypt 2 Science and Humanities College, Shaqra

University, Biology Department, Shaqra,

Postcode 15517, Saudi Arabia * waleed_hamada@yahoo.com + these authors contributed equally to

this work

ABSTRACT

This work is an attempt to overcome antimicrobial resistance problem which dispersed worldwide in particular

developing countries due to misuse of antibiotics. Actinobacteria were isolated and screened against selected

resistant Gram-negative bacteria to detect the powerful antibacterial activity. Identification of the most potent

actinobacterial isolate has been carried out using classical and genetical methods. Antibacterial compound has been

extracted, purified and characterized using accurate and more specific techniques and instruments. Among forty

actinobacterial isolates, only twenty-two isolates could inhibit the growth of Gram-negative bacteria. The most

potent isolate Eg-7 was identified as S. griseorubens, which has a typical 16S rRNA gene. The antibacterial

compound was extracted using ethyl acetate, and separated by High Performance Liquid Chromatography using

methanol and water as a mobile phase. Five active peaks were displayed and retained in the range 40 – 45 min, but

the last three peaks were retained at 41.90, 43.43, and 44.54 min, respectively. The crude extract was analyzed by

liquid chromatography mass spectrum, where the active peak was displayed at 721.325 m/z. The antibacterial

compound was purified using flash column chromatography and gel filtration column chromatography. The active

fraction was analyzed by Infra-Red spectrum, where a broad absorption at 3338 cm-1 was displayed. Molecular

formula of an antibacterial compound was determined by mass spectrum as C35H26N6O4. Nuclear magnetic

resonance analysis was carried out for an antibacterial compound. These results suggest that a new antibacterial

compound that similar quinolone could be produced by S. griseorubens and exhibited a higher activity against

Gram-negative bacteria.

Introduction

The chemical structure of quinolones contains a bicyclic core (Fig. 1) such as nalidixic acid that was discovered in

1960s and used in the treatment of diarrhea and urinary tract infections caused by Shigella spp and Escherichia coli1.

Nalidixic acid is narrow spectrum antibiotic due to low serum concentration, high minimum inhibitory concentration

(MIC) and adverse events2-4. Serious bacterial diseases are completely cured with different generations of

quinolones due to their highly antibacterial activity (Table 1). Fluoroquinolones are improved derivatives of

quinolones due to developed pharmacokinetics which provide a broader spectrum5. Fluoroquinolones like

ciprofloxacin and ofloxacin are the first choice and highly recommended antibiotics due to highly potency, highly

bioavailability, available formulations, highly serum concentrations and mild side effects. Fluoroquinolones inhibit

both Gram-negative and Gram-positive bacteria, so they are widely used in the treatment of tuberculosis, anthraxes

and other serious bacterial diseases6. Rarely, fluoroquinolones affect gastrointestinal tract, nervous system, genome,

and eye7.

Quinolones retards bacterial DNA synthesis via destruction of topoisomerase type II and inhibition of DNA

gyrase and topoisomerase IV, which regulate the chromosomal supercoiling8,9. Mutations in the binding sites of

DNA gyrase and topoisomerase IV, or an acquisition of a resistant plasmid from other sources are the main reasons

for emergence of quinolones resistance by bacteria10-12. Addition of a fluorine atom at R6 position of quinolone

provided a broad-spectrum antibiotic called fluoroquinolone that could inhibit the growth quinolone-resistant

bacteria13. Ciprofloxacin inhibits the growth of Gram-negative bacteria like Pseudomonas spp, while

Page 2 of 16

fluoroquinolones which have a piperazine ring at R7 position and substituent of a cyclopropyl group at the R1 could

inhibit Gram-positive and Gram-negative bacteria14.

Many improved pharmacokinetics of fluoroquinolones are present including high absorption, maximum

serum concentration (Cmax), high efficacy at low doses, and safe output away of renal clearance15, while the output

of quinolones is carrying out by metabolism and renal clearance. Two factors control the efficacy of quinolones;

AUC/MIC and Cmax/MIC, where AUC is the area under the curve. The value of AUC/MIC indicates the efficacy of

quinolone, which be high at >125. The value of Cmax/MIC indicates the antibiotic capacity to overcome a bacterial

resistance, and therefore a high Cmax value over low MIC value is meaning a high antibiotic efficacy, which is

usually > 4.016,17. For example, fourth generation of quinolones is more effective than other generations due to its

highest value of Cmax/MIC18,19. The improvement of quinolones pharmacokinetics is usually performed by

modifications at R5, R6, R7, or R8 positions. The improved quinolones have longer output half-life, better tissue

penetration, increased volume distribution, and better bioavailability. For example, sparfloxacin is an improved

quinolone due to addition of an amino group to R5 position, where the lipophilicity and therefore better tissue

penetration are increased. Also, the addition of fluorine atom to R6 position of quinolone lead to easy penetration to

the bacterial cell and increased the volume of distribution20.

Although quinolones are effective antibiotics particularly fourth generation, some side effects are being

associated, such as gastrointestinal disorders and arthralgia. Therefore, they are prescribing to children more than

adults. Some side effects are accompanied with earlier quinolones due to narrow spectrum activity and low

pharmacokinetics. Prolonged period of quinolone use leads to permanent side effects such as tendon rupture, nerve

damage, and fluoroquinolone-associated disability syndrome. Phototoxicity was observed with quinolones

combination such as combination of clinafloxacin and sparfloxacin. Specific side effects were observed, such as

hematological toxicity with temafloxacin, hepatitis with trovafloxacin, and hypoglycemia with clinafloxacin and

gatifloxacin. Immunological side effects were also observed which represented in the disorders of central nervous

system and genotoxicity, which is usually observed with some fluoroquinolones when exposed to ultraviolet light,

such as lomefloxacin, ciprofloxacin, and moxifloxacin. Although quinolones side effects, they are widely used

because their toxicity is reduced due to structural modifications. Nevertheless, there warmings were introduced by

FDA in 2018s, where fluoroquinolones use is prohibited for patients suffered from aortic aneurysm because they

cause aortic damage21,22.

The proper selection of fluoroquinolone to complete inhibit the pathogenic bacteria is depending on accurate

classification. Four groups of fluoroquinolones are present (Table 2); the first group is only available for oral use

and is widely used in the treatment of urinary tract infections and others. For example, norfloxacin is used in the

treatment of bacterial enteritis, gonorrhoea, and prostatitis, and pefloxacin is mainly used in the treatment of

uncomplicated cystitis. All the members of the second group have in vitro potency against enterobacteriaceae,

Pseudomonas aeruginosa and Haemophilus influenzae, and they exhibit moderate potency against staphylococci,

pneumococci and enterococci; as well as, atypical pathogens like Chlamydia and Mycoplasma. All the members of

the second group are administered in oral or parenteral form, except enoxacin is administered only in oral form and

used in the treatment of urinary tract infections. The second group of fluoroquinolones is used in the treatment of

respiratory tract infections caused by Gram-negative bacteria in particular, skin, soft tissue and bone infections; as

well as, systemic infections including septicaemia. The third and fourth groups differ from the second one, where

the first two have a higher activity against Gram-positive bacteria, such as staphylococci, streptococci, pneumococci

and enterococci. Moreover, they have improved activity against atypical pathogens such as Chlamydia and

Mycoplasma, and in the fourth group also has an improved activity against anaerobes23.

Actinobacteria are Gram-positive bacteria which mainly habitat the soil and they are good producers for a

wide array of secondary metabolites including vitamins, amino acids, nucleotides, and antimicrobial agents.

Quinolones are synthetic-bactericidal agents that inhibit the enzyme topoisomerase II, a DNA gyrase necessary for

the replication of the microorganism. Topoisomerase II enzyme produces a negative supercoil on DNA, permitting

transcription or replication; thus, by inhibiting this enzyme, DNA replication and transcription are blocked. The

polyketides are another class of natural antibiotics synthesized through the decarboxylative condensation of

malonyl-CoA-derived extender units in a process similar to the fatty acid synthesis. The polyketide chains produced

by a minimal polyketide synthase are often further modified (e.g., glycosylated) into bioactive natural products. This

study aims to constrict prevalence and exacerbation of Gram-negative bacteria infections by production of an

effective antibacterial compound from S. griseorubens. This antibacterial compound will be extracted, purified and

characterized to be ready for another study. This work provided a new chemical formula that has very potent

antibacterial activity in particular against Gram-negative bacteria. This chemical formula is similar with that of

quinolone antibiotic. This chemical formula is naturally produced by actinobacteria and may be chemically modified

to introduce a broad spectrum and an effective derivative.

Page 3 of 16

Results

Isolation and screening of actinobacteria

There three soil samples were collected from divergent locations in the farm of National Organization for Drug

Control and Research (NODCAR). The soil which are cultivated in particular is still the mother habitat of

microorganisms especially actinobacteria, which decompose the organic substances within the soil and provide easy

nutrients for plants. Forty unrepeated actinobacterial isolates were isolated on the agar surface of starch nitrate

medium. Each isolate has been assigned with a certain code that was divided into a symbol "Eg" and a serial

number. The antibacterial activity of actinobacterial isolates was detected by screening against test pathogenic

Gram-negative bacteria using agar-disc diffusion method. All clinical researches proved that, Gram-negative

bacteria are more deleterious than Gram-positive bacteria due to different reasons including presence of outer

envelope and biofilm which retard delivering of antibiotics inside the bacterial cell. Gram-negative bacteria are

usually correlated with serious diseases like respiratory system diseases and urogenital tract infections especially if

antibiotics-resistance genes are present labeled either on the chromosomes or extra-chromosomes like plasmids or

transposons. The screening test resulted in presence of twenty-two isolates which produced different levels of an

antibacterial activity. As well known, actinobacteria and Streptomyces spp in particular are characterized by a high

ability for secondary metabolites production including antimicrobial agents, which are widely used in the medical

field. Notably, the levels of an antibacterial activities were relatively similar, except that of the isolate "Eg-7" was

has a highest level of an antibacterial activity, so this isolate was selected as a most potent antibacterial producer

(Table 3).

Identification of actinobacterial isolate

The actinobacterial isolate Eg-7 was identified using classical and gene analysis techniques. According to

International Streptomyces Project (ISP), six agar media were used to determine the cultural characteristics

including growth rate, color of aerial and substrate mycelia, and the color of diffusible pigment (Table 4). The

microbiological media of actinobacteria are rich with nutrients and carbon and nitrogen sources in particular,

because they are consumed by the bacteria to accomplish the required vital physiological processes including

propagation, sporulation, excretion, energy generation, and others. The actinobacterial isolate Eg-7 was found has

gray aerial mycelia on the agar surface of all recommended media, while it was found has a yellow substrate

mycelium on the agar surface of yeast-malt extract (ISP-2), and brown color on the agar surface of the other media.

This isolate was moderately grown on the agar surface of both yeast-malt extract (ISP-2) and inorganic salts starch

(ISP-4) media, while a good growth was shown on the agar surface of oatmeal extract (ISP-3) medium, and a poor

growth was observed on the agar surface of both peptone yeast extract iron (ISP-6) and tyrosine (ISP-7) media.

Although it was found produce a yellow diffusible pigment on the agar surface of both yeast-malt extract (ISP-2)

and oatmeal extract (ISP-3) media, it didn't produce diffusible pigments on the agar surface of the other media.

Physiological and biochemical characteristics of the actinobacterial isolate Eg-7 have been determined (Table

5). The regular morphology of actinobacteria depends on the age of culture, so it must be harvested and prepared at

a proper time to can use by scanning electron microscope. The spiral spore chain was appeared with ellipsoidal

smooth spores (Fig. 2). The gray spore mass was appeared, and the brown and yellow colors were shown for

substrate mycelia and diffusible pigment, respectively. No motility was recorded for this isolate, and the structure of

cell wall was found has LL-diaminopimelic acid, and the sugar pattern didn't be detected. This isolate produced

melanoid pigment and hydrogen sulfide. Xanthin and esculin were recalcitrant substances where they didn't degrade.

The growth of the isolate Eg-7 was not observed at high concentrations of NaCl (10 – 15%). Streptomycin and

amoxicillin antibiotics completely inhibited the growth of the actinobacterial isolate Eg-7. The isolate couldn't grow

at high values of temperature (≥ 40°C) and pH (≥ 10). Apart of amylase, protease and catalase which were produced, other enzymes never produced. Apart of L-arabinose which didn't utilize, other sugars were utilized as a carbon

source. Finally, all amino acids were utilized as a nitrogen source. The sequence analysis of 16S rRNA gene was

performed as a confirmatory identification of the actinobacterial isolate Eg-7. Comparison has been carried out

between the sequence of 16S rRNA gene and reference strains of Streptomycete sequences using DNA BLASTn

(NCBI website). The classical and gene analysis methods proved that, the actinobacterial isolate Eg-7 is belonging

to Streptomyces genera, and S. griseorubens in particular due to a high similarity percentage (99%) between test and

reference strains.

Extraction of antibacterial compound

The antibacterial compound was extracted from the cell free extract of starch nitrate broth medium inoculated with

S. griseorubens using ethyl acetate. The ethyl acetate extract was separated by HPLC using methanol and water as a

mobile phase. The peaks of potential antibacterial compounds were appeared with other peaks of polar and non-

Page 4 of 16

polar impurities (Fig. 3). The last three active peaks were retained at 41.90, 43.43, and 44.54 min, respectively, and

all five active peaks were retained between 40 and 45 min. The largest active peak was dried using N2 gas and its

solubility was tested using acetonitrile, DMSO, methanol, ethanol, and 2-propanol. Apart of ethanol and 2-propanol,

methanol, acetonitrile, and DMSO could solubilize an antibacterial compound. Fraction containing polar compound

displayed more effective antibacterial activity. The solvents affected the extract containing an antibacterial

compound, where polar solvent (water) removed all polar impurities, and non-polar solvent (n-pentane) removed all

non-polar impurities.

Purification, mass spectrum and IR analyses of antibacterial compound

The crude extract of an antibacterial compound was analyzed by liquid chromatography mass spectrum (LC-MS),

which resulted in numerous peaks, but the active one was displayed at m/z 721.325. This crude extract was exposed

to purification process through FCC and GFCC. FCC was perfectly packed with a silica gel 60 as a stationary phase,

and DCM/methanol mixture was used as a mobile phase. The elution and fractionation of the crude extract have

been carried out, and the antibacterial activity was tested with each fraction. The active fractions were pooled and

dried by N2 gas, and then delivered to GFCC which was packed with sephadex LH-20. The elution of the mixture

was carried out by methanol to obtain several fractions. The antibacterial activity was tested with each fraction,

where only four active ones were obtained, pooled and dried using N2 gas. The absorbance of each active fraction

was measured at 254 nm, and the broad active peaks were displayed (Fig. 4). The most active fraction was analyzed

by IR spectrum, which showed a broad absorption at 3338 cm-1 that is characteristic of a hydroxyl group, an

absorption at 3079 cm-1 associated with an alkene, and the absorption bands around 3000 cm-1 that indicated the -C-

H stretches of alkane groups. Absorption at 1728 cm-1 confirms the presence of a carbonyl functional group in the

molecule (Fig. 5). The mass spectrum analysis of an antibacterial compound was performed to determine its

molecular formula, which was recorded as C35H26N6O4.

NMR analysis

Structure determination has been carried out using Bruker BioSpin and Billerica MA for 1D (1H NMR, 13C NMR)

and 2D (HSQC, COSY, TOCSY, and HMBC) extensive NMR spectral analysis. Structure determination of an

unknown compound using homonuclear 1H-1H COSY depends on the ability to detect couplings between

neighboring protons. Correlations between protons and neighboring carbons can be detected using the heteronuclear

HMBC spectrum. Based on the characteristic UV-Vis absorption maxima at 277 and 327 nm; strong IR absorptions

at 3338 cm-1 (-OH), 3079 cm-1 (=CH-); large molecular weight 721.325 Da with a higher number of carbon,

hydrogen, and oxygen atoms from HR-MS and proton signals from 1H NMR, the compound of interest would most

likely be a polyketide. Close inspection of spectral data revealed the presence of minor peaks in NMR spectra,

which could be related to the decomposition of the natural product during the purification process, as well as

distinctive tautomers or conformers. Apart from minor signals, the structure analysis of the unknown polyketide was

started with major peaks present in CD3OD. 1H (Fig. 6) and 13C (Fig. 7) NMR data in combination with the HSQC

analysis showed the presence of 35 carbons attributable to seven (7) sp3 methyl (-CH3) groups among which one

was an oxygenated methyl carbon. There are eleven carbons corresponded to sp2 methine (-CH=C), ten carbons sp3

methylene (-CH2-), eighteen carbons are sp3 methine, and six carbons are quaternary carbons. 13C-NMR

interpretation disclosed three sets of distinct chemical shifts; saturated methyl (-CH3), methylene (-CH2), and

methine (sp3 CH).

Discussion

Streptomycetes can survive in a wide array of environments including the soil, water, air, wastes, and even on the

solid surfaces. Moreover, they can live under mild and extreme conditions such as hot, cold and salty environments.

This unique ability of actinobacteria is due to various factors including sporulation profile, utilization of a wide

array of carbon sources and production of antimicrobial agents. In addition, a broad metabolic profile of

streptomycetes is due to presence of large genome containing hundreds of transcription factors that control gene

expression, allowing them to respond to specific needs24. The most potent antibiotic producer is Streptomyces genus

so far. Apart of antibiotics, different bioactive compounds were produced by Streptomyces and utilized as effective

pharmaceutical products including anticancer and immunosuppressant compounds25.

The most potent antifungal actinobacterial isolate was identified as S. griseorubens using phenotypic and

molecular methods. The morphology was found appear as branched substrate and aerial mycelia which carried

smooth-surfaced ellipsoidal spores in open hooked spore chains. The cell wall contained LL-diaminopimelic acid

and the sugar pattern didn't detect. A grayish aerial spore mass was appeared on 7 standard media recommended for

Page 5 of 16

the genus Streptomyces. The identification was confirmed by analysis of 16S rRNA gene sequence, which was

found has 1096bp26.

1-butanol was the first-choice organic solvent for extraction of an antibacterial compound from the filtrate of

Rhodococcus sp. by HPLC due to low solubility and immiscibility in an aqueous solution. Therefore, 1-butanol is

considered one of effective alcoholic solvents used for an extraction of antimicrobial compounds. The antibacterial

compound was extracted by 1-butanol from both wild and mutant strains, and the antibacterial activity of extracted

antibacterial compound from mutant strain was tested using agar-disc diffusion method compared with that of wild

strain. UV-Vis spectroscopy analysis was carried out before fractionation process27.

According to the analysis of IR spectrum of an antibacterial compound that is identified in the later as

quinolone, the active peaks elucidated at the range 1735–1707 cm-1 due to the vibration of carboxylic group, while

the intense one elucidated at 1617 cm-1 which assigned as v(C=O)p, where p is pyridone. The peak which displayed

at 3400–3200 cm-1 assigned to O–H stretching vibrations. Two characteristic peaks at 1556 and 1494 cm-1 that could

be assigned as m(O–C–O) asymmetric and symmetric stretching vibrations; respectively, moreover, it is very

difficult to assign other vibrations in the spectra of ciprofloxacin due to the presence of other numerous peaks could

be present in these regions such as the presence of C=C stretching vibration of the aromatic rings around 1620 cm-1

and the CH bending vibrations (m-CH) in the region between 1440 and 1550 cm-1 and the stretching vibration of the

quinolone ring system (m ring) around 1400 cm-1 28. The mass spectrum of quinolone was performed using ESI-

MS/MS technique which was used in characterization of several compounds. The signal appears at 331 m/z refers to

appearance of the molecular formula [C17H19FN3O3]+ which is high stable29. The absorption spectrum of quinolone

was determined at 0.25 × 10-5 mol/L in ethanol. The characteristic absorption peak was displayed at 322 nm which

could be due to n–p* (HOMO–LUMO) electronic transition. The second active peak with high absorbance was

displayed at 275 nm. Unsaturated hydrocarbons attached to O2 atom are characterized by these transitions30.

The internal hydrogen atoms of quinolone structure link between the hydroxyl residue of the carboxyl group

and the carbonyl residue of the pyridone nucleus to affect the chemical shifts of C-4 and CO signals, which are

shifted downfield. The implementation of the substituent on nitrogen changed the carbon chemical shift in the 2-

position. For all carbons of the benzene ring, incremental chemical shifts elicited by the presence of the 3-

substituted 4-quinolone ring, therefore similar data pertinent to the fused triazole or pyrazine ring, allow the

prediction of chemical shifts in various fused multi-ring systems31.

Methods

Sampling of soils and isolation of actinobacteria. Three soil samples were collected from divergent locations in

the farm of National Organization for Drug Control and research (NODCAR). The soil sampling was accomplished

at depth 10 cm to avoid the arid surface that is usually free from microorganisms. The soil sample was directly

placed in a sterile plastic bag and fast transported to the laboratory for air drying. According to Johnson et al.32, 10 g

of air-dried soil were suspended in 100 ml of sterile distilled water for 20 min to make a soil suspension, which has

been filtered through sterile cotton piece to obtain a semi-clear soil extract. Soil extract was diluted using serial

dilution method (10-1 to 10-8). One ml was withdrawn from each fraction, and then pipetted and streaked by sterile

glass rod on the agar surface of starch nitrate agar medium33. All plates were incubated at inverted position for 7

days at 30°C. After incubation period and by visual examination, all separated actinobacterial colonies were picked

up independently by a sterile platinum loop, and then streaked on the agar surface of starch nitrate agar medium to

obtain purified actinobacterial isolates, which were preserved on agar surface of starch nitrate slants at 4°C for one

month and subcultured regularly. Duplication of plates has been performed.

Test pathogenic bacteria. American Type Culture Collection Laboratory (ATCC) was the sole source of test

pathogenic Gram-negative bacteria. Five strains were obtained named Acinetobacter lwoffi ATCC 17925, E. coli

ATCC 25922, Klebsiella pneumoniae ATCC 33495, P. aeruginosa ATCC 35032, and Enterobacter cloacae ATCC

35030.

Antibacterial test. The antibacterial activities of actinobacterial isolates were detected by screening them against

pathogenic Gram-negative bacteria. The pathogenic bacterial strains were prepared by culturing them in soybean-

casein dextrose broth medium, which has been incubated for 24 h at 35 ± 2°C. Actinobacterial discs of 7 days old

were placed on the agar surface of seeded soybean-casein dextrose agar plates with pathogenic bacterial strains, and

all plates were incubated for 24 h at 30°C. Examination of plates has been carried out visually to determine the

antibacterial activities represented by appearance of an inhibition zones around active actinobacterial discs.

Page 6 of 16

Identification of the most potent antibacterial isolate of actinobacteria. Identification of the most potent

antibacterial isolate of actinobacteria was performed according to Shirling and Gottlieb34, where morphological,

cultural and physiological characteristics were determined. Confirmatory identification was carried out by analysis

of 16S rRNA gene sequence. The DNA was extracted according to Sambrook et al.35, and the concerned gene was

amplified by a thermocycler (Cetus Model 480; PerkinElmer, Waltham, MA, USA) using the universal primers 27f

(5′-AGA GTT TGATCC TGG CTC AG -3′) and 1525r (5′-AAG GAG GTG ATC CAG CC-3′) at 94°C for 5 min, 35 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 90 s, and a final extension step at 72°C for 5 min. The product

was sequenced by a BigDye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA, USA) on an

ABI 310 automated DNA sequencer (Applied Biosystems). Homology of the 16S rRNA sequence was analyzed

using the BLAST algorithm that is an available in GenBank.

Extraction of an antibacterial agent. Starch nitrate broth (1500 ml) was prepared and distributed into 3 flasks (1

L), where each one was contained 500 ml. Each flask was inoculated with one ml of actinobacterial suspension

(most potent). All flasks were incubated in a shaking incubator at 20°C and 120 rpm for 14 days. After incubation

period, the actinobacterial suspensions of three flasks were pooled and supplemented with 300 ml of 1-butanol

because it is an immiscible with an aqueous solution. This mixture was incubated in a shaking-incubator at 20°C and

120 rpm for 1 h. After incubation period, the actinobacterial suspension was centrifuged at 6000 rpm for 10 min, and

the supernatant was separated using a separating funnel, the top organic layer (1-butanol) was collected into a

beaker, and both layers (organic and the aqueous) tested for activity to ensure the all of the compounds had been

extracted from the broth culture. The butanol extract was evaporated using a rotor evaporator at 25°C36.

Antibacterial activity test. The antibacterial activity of an extract was tested against pathogenic Gram-negative

bacteria using agar-disc diffusion method37. A loopful of a single bacterial colony was immersed in 2 mL of

soybean-casein dextrose broth. All test tubes were incubated in water bath with shaking at 27°C for 18 h. The sterile

filter paper disk was saturated with an extract and placed on an agar surface of bacterial seeded plates, which were

incubated for 24 h at 35 ± 2°C. Appearance of an inhibition zone around the disk refers to an antibacterial activity

and vice versa.

Flash column chromatography (FCC). The first separation step of an antibacterial agent was performed by FCC

that was packed with a silica gel 60 as a stationary phase38,39. The separation by FCC depends on the partitioning

between silica gel 60 (stationary phase) and dichloromethane (DCM) (mobile phase), which migrated under low

pressure. Different compounds including an antibacterial compound in the crude mixture have different affinities

with the stationary phase due to a chemical charge or an adsorption operability40,41. FCC was sealed at the bottom

with a piece of cotton-wool and a small layer of sand (1-2 cm), and then the silica gel 60 was gently poured until

successful packing has been accomplished. Vacuolation was done through the stopcock of the column to condense

the silica gel and to obtain a tight packing. Sodium sulfate solution was poured on the top of the column to prevent

charges exhaustion. DCM was gently poured with vacuolation onto the column until solvents elution carried out.

The dried butanol extract was mixed with silica gel 60 and a low amount of DCM. This mixture was passed through

a stationary phase with methanol as polar solvent and DCM as non-polar solvent. Different ratios of DCM and

methanol (v/v) were elucidated as shown in (Table 6). The fractionation has been carried out (10 mL/fraction), and

the absorbance of each one was measured at 254 nm by using UV-spectrophotometer. The antibacterial activity was

detected for each fraction by using the agar-disk diffusion method. The active fractions were pooled and let stand

under fume hood to evaporate the solvent and to dry the mixture, which was dissolved in 2-propanol and stored at

4°C for further purification.

Gel filtration column chromatography (GFCC). The mixture of FCC was transmitted to GFCC, which depends

on the size of molecules in separation process. The large molecules never face any retardation during their trip of

migration through stationary phase, because the big size of them don't allow to enter the pores of stationary phase

and therefore they move easily away the resin under pressure of mobile phase. On contrary, the small size of some

molecules enables them to enter inside the molecules of the resin through the pores, and therefore they be retarded

and eluted slowly, so small molecules can be picked up at the last fractions. Sephadex™ LH-20 was used as a resin

of the column, and it was prepared by hydroxypropylation of sephadex G-25, which has hydrophilic and lipophilic

characteristics, so it swells in aqueous solutions and organic solvents. Sephadex™ LH-20 dry powder (28.78 g) was

placed in a beaker (250 ml), and supplemented with methanol gradually with gently stirring until be swollen during

2 h. The beaker was shaken every 30 min to remove any air bubbles trapped in the medium. The sephadex was

poured gently inside the glass column chromatography where the column was filled evenly without forming of air

bubbles. The solvent was added and passed through the resin under atmospheric pressure. The solvent was

Page 7 of 16

withdrawn through the opened stopcock of the column until the column was tightly packed, and then the stopcock

was closed. The dried 1-butanol extract was dissolved in a low amount of isopropanol and passed through a

sephadex and eluted with methanol as a mobile phase. The fractions were collected and tested for antibacterial

activity. The absorbance of each fraction was measured by UV-spectrophotometer at 254 nm. The active fractions

were pooled and let stand under fume hood to be dried. The dried extract was dissolved in a low amount of 2-

propanol to be prepared for high-performance liquid chromatography (HPLC) analysis42.

HPLC analysis. The antibacterial extract was purified by HPLC (LC-10AS), which was equipped with 2 solvents;

A and B. Two pumps A and B were used to pump the mobile phase to generate a maximum pressure of 6000 psi

(lb/in.2) or 414 bar43,44. Mobile phase degassing inside the pump was mainly used to prevent formation of the air

bubbles, which cause a problem in the solvent delivery and forms specious peaks in the output by the detector45. The

solvents were placed inside HPLC with a stir bar, which was connected to a vacuum pump by the stopper. HPLC

was equipped with UV-Vis detector (SPD-10A)46.

Characterization of an antibacterial agent. The antibacterial compound was characterized according to Butler47.

Stepwise of characterization has been achieved to detect and elucidate the identity of an antibacterial compound.

UV-Vis spectroscopy, IR spectroscopy, mass spectroscopy, and NMR (1D and 2D) were used as urgent tools for

chemical formula elucidation.

Ultraviolet-Visible Spectroscopy (UV-Vis). UV-Spectra (280 – 400 nm) of all fractions of both FCC and GFCC

were measured by UV-Vis spectrophotometer (Carey 8454), and methanol was used as a blank. The purified

compound of HPLC was dissolved in acetonitrile and its absorbance was measured at 254 nm, and acetonitrile was

used as a blank47.

Infrared spectroscopy (IR). IR-spectra were measured in KBr pellets with a spectral range of 6,000-350 cm-1 using

a genesis II FTIR spectrometer47.

Liquid chromatography-mass spectroscopy (LC-MS). The crude extract of the most potent actinobacterial isolate

was exposed to LC-MS analysis. The analysis was done using the gradient elution method with solvents A and B

containing 0.1% aqueous formic acid and acetonitrile, respectively. The program started with 30% B which was

increased to 40% B from 0-5 min. After 5 to 10 min it was increased to 50% B; from 10 to 15 min it increased to

60% B; from 15 to 20 min it increased to 80% B; from 20 to 25 min increased to 100% B, which was maintained

from 25 to 35 min, at a flow rate 1.0 ml/min. From 35 to 35.5 min 100% B was dramatically decreased back to 30%

B, which was kept from 35.5 to 40 min, to reconstitute the column before the next run. The column oven

temperature was maintained at 25°C, and the temperature of the autosampler was set to 4°C. An electrospray

ionization probe was used to ionize the sample at a 3.5 kV spray voltage. The sheath gas flow was set to 50 units

and the auxiliary gas was set to 25 units. The S-lens level was set to 50 units. The conditions were kept constant for

positive ionization mode acquisition. For complete scan profiling experiments, the MS was run with a resolution of

140,000 and with a scan range of 80-1000 m/z, for all ion fragmentation (AIF) scans. The resolution was 140,000

with a scan range of 80-1000 m/z, with a normalized collision energy (NCE) of 20 eV. Xcalibur software was used

to examine the results47.

High-resolution mass spectrometry (MS). The high-resolution mass analysis was carried out on Bruker maXis II

mass spectrometer. The sample was ionized using electrospray ionization in positive (ESI+) mode. The sample was

dissolved in an acetonitrile and water mixture (50:50) with 0.1% formic acid47.

Nuclear magnetic resonance (NMR) spectroscopy. 1H-NMR, 13C-NMR, and 2D-NMR experiments were

performed on a Bruker Avance II 600 MHz NMR spectrometer (1H 600 MHz; 13C 150 MHz). It was equipped with

5 mm probe using deuterated methanol as a solvent. All NMR experiments were carried out at room temperature.

Chemical shift values were measured in parts per million (δ, ppm). The coupling constants value (J) was described in Hz. The splitting patterns of proton signals were also designated as follows: singlet (s), doublet (d), a doublet of

doublets (dd), a doublet of the doublet of doublets (ddd), triplet (t), the quartet (q), and the multiplet (m)47.

Page 8 of 16

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Acknowledgements

Authors sincerely thank Head and members of Microbiology Department for their support to this work, and also

deeply grateful to Head and members of Biology Department for providing the necessary support to can complete

this work.

Author contributions statement

W. M. provided the scientific idea, and performed collection of soil samples, isolation and identification of

actinobacterial isolate, antibacterial test, and writing and correspondence of a manuscript for publication. M. A.

brought-in the bacterial strains, and performed extraction, purification and characterization of an antibacterial

compound.

Additional information

Correspondence and requests for materials should be addressed to Waleed M. Abdulkhair

The corresponding author is responsible for submitting a competing interests statement on behalf of all authors of the

paper.

Figure 1. Core structure of quinolone antibiotics

Page 11 of 16

Figure 2. Scanning electron micrograph of the actinobacterial isolate Eg-7

Figure 3. HPLC chromatogram of an extract of S. griseorubens in methanol

Page 12 of 16

Figure 4. UV spectra of active fractions of sephadex LH-20 column eluted with methanol

Figure 5. IR spectrum of an antibacterial compound

Page 13 of 16

Figure 6. 1H NMR spectra of an antibacterial compound, (A) C1, (B) C2, (C) C3

Page 14 of 16

Figure 7. 13C NMR spectra of an antibacterial compound, (A) D1, (B) D2, (C) D3

Page 15 of 16

Generation Name Spectrum activity

1 Nalidixic acid Gram-negative bacteria

(except Pseudomonas spp.)

2a Enoxacin, norfloxacin and

ciprofloxacin

Gram-negative bacteria, M. pneumonia and

C. pneumonia

2b Ofloxacin and lomefloxacin Gram-negative bacteria and S. aureus

3 Sparfloxacin, grepafloxacin,

clinafloxacin and gatifloxacin

Gram-positive bacteria

4 Moxifloxacin, gemifloxacin,

trovafloxacin and garenoxacin

Aerobic and anaerobic Gram-positive and Gram-negative bacteria

Table 1. Different generations of quinolones and their spectrum activities

Group Description Members

1st Oral forms treat urinary tract infections Norfloxacin and pefloxacin

2nd Oral and parenteral forms treat systemic

indications

Enoxacin, fleroxacin, ofloxacin and ciprofloxacin

3rd Oral and parenteral forms treat

Gram-positive and atypical pathogens.

Levofloxacin, sparfloxacin and grepafloxacin

5th Oral and parenteral forms treat

Gram-positive, atypical pathogens and

anaerobes.

Gatifloxacin, trovafloxacin, moxifloxacin and

clinafloxacin

Table 2. Classification of fluoroquinolones

Isolate

(Eg)

Antibacterial activity measurement by an inhibition zone (mm)

A. lwoffi E. coli K. pneumoniae P. aeruginosa E. cloacae

5 & 6 25.0 30.0 25.0 22.0 20.0

7 42.0 45.0 35.0 30.0 45.0

11 20.0 25.0 20.0 25.0 20.0

15 20.0 20.0 25.0 30.0 25.0

16 25.0 27.0 20.0 27.0 25.0

17 & 18 25.0 30.0 20.0 25.0 30.0

20 20.0 20.0 25.0 27.0 20.0

22 & 23 25.0 20.0 25.0 30.0 20.0

25 30.0 25.0 20.0 25.0 25.0

26 30.0 20.0 20.0 20.0 25.0

27 25.0 27.0 20.0 30.0 25.0

30 25.0 27.0 20.0 25.0 20.0

32 25.0 20.0 25.0 25.0 20.0

33 20.0 25.0 25.0 20.0 27.0

34 & 36 20.0 20.0 20.0 20.0 27.0

37 20.0 30.0 25.0 20.0 20.0

38 20.0 25.0 25.0 20.0 30.0

40 20.0 25.0 25.0 27.0 30.0

Table 3. Antibacterial activities of active actinobacterial isolates

Page 16 of 16

Agar media Growth Color

AM SM DP

Yeast-malt extract ISP-2 Moderate Gray Yellow Yellow

Oatmeal extract ISP-3 Good Gray Brown Yellow

Inorganic salts starch ISP-4 Moderate Gray Brown None

Glycerol asparagine ISP-5 Good Gray Brown None

Peptone yeast extract iron ISP-6 Poor Gray Brown None

Tyrosine ISP-7 Poor Gray Brown None

AM, Aerial Mycelia; SM, Substrate Mycelia, DP, Diffusible Pigment

Table 4. Cultural characteristics of an actinobacterial isolate Eg-7

Parameter Test Result

Morphological

characteristics

Shape of spore chain Spiral

Spore shape ellipsoidal

Spore surface Smooth

Motility Non-motile

Cell wall hydrolysis Diaminopimelic acid (DAP) LL-DAP

Sugar pattern Not detected

Physiological

characteristics

Melanoid and H2Sproduction Positive

Xanthin and esculin degradation Negative

Tolerance of NaCl (10-15%) Negative

Streptomycin and amoxicillin resistance Negative

Growth at ≥ 40°C Negative

Growth at ≥ 10 pH Negative

Enzymatic activity

Amylase, protease and catalase production Positive

Lipase, cellulase and coagulase production Negative

Nitrate reductase and urease production Negative

Utilization of different

carbon sources

Glucose, fructose, galactose and sucrose Positive

Raffinose, rhamnose and mannitol Positive

Arabinose Negative

Utilization of different

nitrogen sources

Cystiene, valine, alanine, leucine and histidin Positive

Lysine, tyrosine and phenylalanine Positive

Table 5. Physiological characteristics of an actinobacterial isolate Eg-7

DCM (volume in ml) Methanol (volume in ml) DCM-Methanol ratio

300 0 1:0

300 10 30:1

300 20 15:1

300 30 10:1

250 50 5:1

150 150 1:1

0 300 0:1

Table 6. DCM and methanol ratios used in flash column chromatography

Figures

Figure 1

Core structure of quinolone antibiotics

Figure 2

Scanning electron micrograph of the actinobacterial isolate Eg-7

Figure 3

HPLC chromatogram of an extract of S. griseorubens in methanol

Figure 4

UV spectra of active fractions of sephadex LH-20 column eluted with methanol

Figure 5

IR spectrum of an antibacterial compound

Figure 6

1H NMR spectra of an antibacterial compound, (A) C1, (B) C2, (C) C3

Figure 7

13C NMR spectra of an antibacterial compound, (A) D1, (B) D2, (C) D3